Control of protein tyrosyl phosphorylation is an important mechanism for the regulation of cellular proliferation, the cell cycle, differentiation, and cell death. Disregulation of these control mechanisms can lead to neoplasia. Although much has been learned about how protein tyrosine kinases (PTK) help determine the steady state level of cellular phosphotyrosine, little is known about how phosphotyrosine phosphatases (PTPs) contribute to this process, and virtually nothing is known about how PTPs might contribute to disease. We will elucidate the biological roles of two nontransmembrane PTPs, PTP1B and SHPTP2, cloned during the first four years of this grant. Initial studies have implicated PTP1B in control of the cell cycle and in cell death pathways. In cycling cells, PTP1B resides in a high molecular weight complex on the cytosolic surface of the endoplasmic reticulum, and undergoes cell cycle- dependent modifications, including alternative splicing during G0 and G1, and new serine phosphorylation at G2/M. However, in human keratinocytes induced to undergo a terminal differentiation/cell death pathway by calcium ionophores, PTP1B is cleaved and released as an active fragment into the cytosol. This is reminiscent of a bacterial virulence determinant that kills cells by dephosphorylating tyrosol phosphorylated suggesting that PTP1B cleavage is important in keratinocyte cell death pathways. SHPTP2, the apparent human homologue of Drosophila csw, interacts directly with several ligand-activated growth factor receptor and becomes tyrosyl phosphorylated in response to growth factor addition, suggesting that it plays a critical role in early growth factor response. In this grant, neutralizing monoclonal antibodies against PTP1B will be generated to allow PTP1B mitotic phosphorylation sites will be mapped, and the biologic role of PTP1B in mitosis explored by microinjection of neutralizing antibodies and transient overexpression of wild type and phosphorylation site mutant in mammalian cells and S. pombe. The precise cleavage site of PTP1B in ionophore-treated keratinocytes will be defined and the role of PTP1B cleavage in inducing cell death assessed in normal cells and in skin cancers, which are refractory to induction of cell death pathways. The precise sites of interaction of SHPTP2 with growth factor receptors will be determined, and the effects of receptor binding and tyrosyl phosphorylation on SHPTP2 enzymatic activity assessed. PDGF receptor mutants that fail to bind SHPTP2 will be used to identify SHPTP2 downstream signaling targets. In collaborative studies, the ability to SHPTP2 to complement csw mutations will be determined, and if complementation is obtained, suppressor and potentiator screens in Drosophila will be used to suggest other components of SHPTP2 signal transduction pathways, Finally, a rapid in vitro bioassay for mapping SHPTP2 functional domains and targets will be developed using FGF-induced mesoderm induction in Xenopus as a model system. Since PTP1B and SHPTP2 appear to participate in control of all of the processes--the response to growth factors, cell cycle control, control of differentiation, and control of cell death--that are eluded by cancer cells, the results of the proposed studies should yield critical information into how PTPs help control these processes. Moreover, since tyrosyl phosphorylation is so commonly disregulated in cancer cells, this work should have important implication for understanding mechanisms of oncogenesis, and may suggest new therapeutic approaches to combat neoplasia.